1. Field of the Invention
The present invention relates to a process of preparing relaxin, and more particularly to a process of preparing relaxin of high purity from milk, as well as relaxin prepared in accordance with such processes, and pharmaceutical products containing such relaxin of milk origin.
Relaxin was first described in 1926 by Frederick Hisaw who designated it as a hormone. He discovered the hormone when injecting blood serum, extracted from pregnant rabbits, in guinea-pigs and observing a relaxation of the symphyseal ligament.
The further elucidation of relaxin in subsequent years was rather scarce, and only at the beginning of the seventies did Sherwood and O'Byrne succeed in obtaining relaxin of high purity by column-chromatographical methods. The purification methods of Sherwood et al. [O. D. Sherwood and E. M. O'Byrne: Arch. Biochem. Biophys. 160, 185 (1974)], to this date, are the most frequently employed ones, as well as being the most effective methods for the preparation of relaxin of high purity.
In subsequent developments, relaxin was not only determined in the organs of the genital tract, but also in the blood serum of various species of mammals (homo sapiens, cattle, pig, dog, rat and mouse), primarily during the pregnancy period thereof. The primary locus of production of the hormone is presumably the corpus luteum of the ovary, and relaxin is there prepared together with estrogen, progesterone and androgen. Isolation of relaxin was primarily from the ovaries of pregnant pigs, due to the considerably greater concentration of relaxin in comparison to other
The relaxin from pigs and sharks, aside from relaxin from rats, is to date the only of the hormones of the type in which the primary structure is known by sequencing of the isolated peptide. On the other hand, relaxin was not only determined in the females, but also in male mammals (human), as well as in male (chicken) and female non-mammals (shark).
In the human and chicken the relaxin was found in the testes. It is of note, however, that relaxin is not evident in the testicles or blood serum of bears or rats. Relaxin increases the sperm immobility in male species.
The concentration of relaxin increases strongly during the course of pregnancy of the animals. Very frequently an increase in the concentration occurs just prior to giving birth.
It is presumed that the corpus luteum is the primary source of relaxin in women, particularly during pregnancy where it is determinable in the blood. The formation of relaxin is so small in females which are not pregnant that the determination in the blood is hardly possible. The hormone hitherto only presumed in women was recently also determined in human semen or seed plasma.
The content of relaxin-containing substance in human milk and in blood plasma, at various time periods after giving birth, is indicated in the following table.
TABLE ______________________________________ Number of Milk pg/ml Plasma pg/ml Period Patients (Range) (Range) after Birth ______________________________________ 6 193 (n.n.-700) 82 (n.n.-203) day 3 11 286 (n.n.-617) 28 (n.n.-143) day 4 11 391 (n.n.-790) 19 (n.n.-150) day 5 7 438 (n.n.-810) 32 (n.n.-223) day 6 6 568 (353-930) n.n. 2-4 weeks 8 464 (177-741) n.n. 5-12 weeks 4 495 (229-937) n.n. 18-34 weeks ______________________________________ n.n. = not detectable.
It is known that relaxin is produced from corpus luteum during the pregnancy of many animals. For many years it was not known whether the placenta is another rawmaterial source for the production of human relaxin [M. X. Zarrow, E. G. Holmstrom and H. A. Salhanick: J. Clin. Endocr. 15, 22 (1955); F. D. Dallenbach and G. Dallenbach-Hellweg: Virchows Arch. Path. Anat. Physiol. 337, 301 (1964 ) and G. Weiss, E. M. O'Byrne and B. C. Steinetz: Science 194, 948 (1970)]. It is now appears that the human decidua contains relaxin [M. Bigazzi, F. Nardi, P. Bruni and G. Petrucci: J. Clin. Endocrinol. Metab. 51, 939 (1980); S. Yamamoto, S. C. M. Kwok, F. C. Greenwood and G. D. Bryant-Greenwood: J. Clin. Endocrinol. Metab. 52, 601 (1981); S. Krassnigg, H. K. Rjosk, G. K. Stalla and K. von Werden: Acta Endocrinol. Suppl. 246, 99 (1982)].
More recent investigations have shown that physiological effects are attributable to relaxin, particularly human relaxin, and, accordingly, it is nowadays of considerable clinical interest.
Relaxin exhibits effects on the uterus, for example, it affects the uterus motility and causes a softening of the cervix. In vitro and in vivo investigations of uteri of rats and mice have shown a prevention of spontaneous contractions of the myometrium through relaxin. Many biochemical changes occurring in the cervix are induced by relaxin. This is the case in the intake of water, glycogen, the depolymerization of the base-substance, and the activation of the collagen-peptidase.
Relaxin, furthermore, shows effects on the vaginal epithelium, since it brings about hardening, and shows an effect on the growth of breast glands. Relaxin acts synergistically with progesteron and estrogen and produces an increase of the lubulo-alveolar tissue. It furthermore causes dilation of the symphyseal ligament.
Distinctions are made between relaxins of different origins, i.e. from pigs, cattle, rats, depending wherefrom the respective substance was won. The different relaxins are all polypeptide hormones which vary, in part and in minor aspects, in their amino acid sequence, the molecular weight, and their relaxin activity.
However, two peptide chains are common in relaxin, which chains are joined by two cystin radicals. Furthermore, all relaxins show similar pharmacological effects.
2. Description of the Prior Art
A process for the extraction and purification of relaxin from ovaries of pregnant animals has been described in U.S. Pat. No. 2,952,431. A process of preparing human relaxin from foetus-membranes is described in DE-OS No. 3,102,487 (DE-OS=German Layed Open Patent Publication). These known processes, however, entail the disadvantage that starting materials are used which are difficult to obtain and are, furthermore, available only in limited quantity. Furthermore, the isolation and purification includes many steps and, accordingly, such known processes are labor-intensive and very expensive. The report "Investigations of Purification, Determination and Effects of Relaxin (Untersuchungen uber Reinigung, Bestimmung und Wirkung des Relaxin)," by H. Struck, Forschungsberichte des Landes Nordrheinwestfalen (Research Reports of the State of North-Rhine-Westfalia) No. 2304, provides details for purification of relaxin, its determination, and its effects.
Further details in purification and determination, or identification, of relaxin may be had from the recently published summary, Gillian D. Bryant-Greenwood: Endocrine Reviews, Vol. 3, No. 1, 1982, p. 62, as well as the references cited therein. This summary and the references mentioned therein are expressis verbis referenced herein.
A new purification process for relaxin, including among others, gel-permeation and ion-exchange chromatography, has also recently been described, M. J. Fields et al.: Annals of the New York Academy of Sciences, Vol. 380, 1982, p. 36 ff.
To-date, however, there was a lack of opportunities to recover relaxin in larger quantities and in a simple manner. As indicated in the foregoing, the starting materials for isolating relaxin are relatively scarce and,. accordingly, relaxin is accessible only to a very limited extent. The full synthesis of relaxin could not be achieved and it can be presumed that relaxin prepared synthetically would be rather expensive.
The known methods of preparing relaxin yield a product having pharmacological effects which vary in conformity with the particular method of preparing, as well as being subject to the properties or effects of the starting material for preparing relaxin. This gives rise to the assumption that relaxin identified and obtained in accordance with the known methods is not of constant uniformity, and moreover also contains impurities which could not be eliminated in the past.